Ferritin, a metalloprotein which stores iron in a bioavailable form, overcomes the low solubility (10-18M) of iron in living systems. Small amounts of ferritin occur in all cells of higher organisms, providing iron for proteins of DNA synthesis, electron transport, and oxygen activation and transport, while large amounts occur in specialized cells of iron storage, e.g. liver, spleen, and red cells of embryo. Pathological conditions occur when iron stores are low or are overloaded. Superimposed on a highly conserved sequence are cell-specific features of ferritin sequence and structure which may relate to function and/or regulation. Ferritin structure could be further modified by cytoplasmic components leading to functional changes. We have recently identified such a change: ferritin subunit dimer crosslinks, a posttranslational modification of ferritin, appear to regulate the iron content of ferritin in vivo. The observation is the first identified, natural structural modification related to the iron storage function of ferritin, to our knowledge. Crosslinking with F2DNB replicates the effect. The structural requirements for crosslinks (which are not -S-S) will be examined in terms of the identity of the linked amino acids, the sequence and subunit restrictions (by sequence analysis and comparison to known subunit sequences), the sequences recognized by monoclonal antibodies sensitive to the presence of crosslinks, the biosynthetic pathway of crosslink formation (H-3-leucine pulse labeling) and cytoplasmic components which may influence crosslinks (transpeptidases? ascorbate?). Cloned ferritin cDNA from lamb spleen or other sources will be prepared and used to gain information about the sequence and regulation of ferritin mRNA related to crosslinks. The functional effect of crosslinks will be measured as the effect of crosslink-sensitive monoclonal antibodies on iron uptake and release in vitro and as the effect of crosslinks on Fe-protein interactions, phosphate-Fe-protein interactions, and iron core structure, using X-ray absorption spectroscopy (EXAFS and XANES). The results will be important in understanding the molecular basis for pathologically altered iron storage, e.g. iron deficiency anemia, hemochromatosis, thalassemia, and cirrhosis, as well as understanding the relationship among cell-specific features of protein structure, cytoplasmic modifying agents, and regulation of function.